The overall goal of this program is to increase our understanding of the cellular and molecular events controlling the formation of connective tissue during wound repair. Specifically, the experiments described here are designed to identify the cytokines which are present at sites of wound repair which control the migration and growth of connective tissue that identify the cell types which are producing these factors; and identify and study the mechanisms which regulates the production of these factors and their action on target cells. The first aspect of this program consists of experiments designed to determine the physiological role of a new family of cytokines termed Leukocyte-derived growth factors (LDGF's). These peptides range is size from 6-14 kD, are members of the CXC (KC/gro) gene family of cytokines. Some of these peptides exhibit PDGF-like biological activity and appear to act via the PDGF alpha and beta receptors while the proteolytic fragments of the same peptide looses this activity and become a neutrophil activating peptide (NAPs). Because a single gene appears to encode peptides which can be processed to act as a connective tissue cell growth factor or a neutrophil chemoattractant the regulation of the production of these peptides could play a central role in the "Regenerative" or "Degradative" pathways of inflammation. Specific cDNA probes for the LDGF transcripts will be used to study the expression of the LDGF gene in isolated leucocytes in culture and in wound tissue and to clone the human LDGF gene. The nucleotide sequence of the gene will be determined as well as its overall structure and chromosomal location in order to develop a foundation of information to more precisely understand the molecular mechanisms governing the regulation of this gene during normal and pathological states. Specific antibodies directed against the regions of the LDGF peptides will be prepared and used to study the synthesis and processing of this peptide and its related products in isolated leucocytes and in wound tissue. Molecular and cell biological techniques will be used to produce recombinant LDGFs to study the structure and function of these molecules. Biological and biochemical methods will be used to compare the action of LDGF with PDGF in both in vitro and in vivo systems. Because many of the fibrotic disorders are prefaced by chronic inflammation it is likely that these same mechanisms may act to control connective tissue formation during the pathological process and a greater understanding of the regulation of the production and action of these cytokines may lead to new insights for the control and treatment of these maladies. The second aspect of this program is to address the growth regulation of wound- derived connective tissue cells. The response of these cells to various growth factors including PDGF and LDGF will be studied. Also, the expression and function of PDGF receptors in the wound-derived connective tissue cells will be studied in both cultured cell and in wound tissue. Preliminary data indicates that fibroblastic cells isolated from wound tissue at different stages of the repair response exhibit very different growth potentials in vitro. Studies will be initiated to determine the basis for this difference in growth potential. The response of the cells to identified growth factors will be characterized as well as the potential production of autocrine growth stimulators or inhibitors examined. Additionally, the basal level of expression of various growth regulatory proto-oncogenes will be examined to determine if there is any correlation of overexpression of these genes with the increased growth potential of these cells. These studies could lead to the biochemical identification of differences among fibroblastic cells that lead to the overgrowth of these cells in the scars that occur after burn injury.